PROJECT SUMMARY In response to funding opportunity announcement PAR-16-176, this joint proposal by Drs. Christoph Rader from The Scripps Research Institute (Jupiter, FL) and Natarajan Muthusamy from The Ohio State University (Columbus, OH) proposes to validate a novel target for immunotherapy of chronic lymphocytic leukemia (CLL). As part of the proposed study, we will develop novel T-cell engaging bispecific antibodies (biAbs) and a novel transgenic mouse model to assess and initiate further preclinical development. Our research team will generate, validate, and deliver novel biAbs that are specifically designed to recruit and activate T cells for selective and potent eradication of malignant B cells in CLL patients. CLL is the most common leukemia in the U.S. with a lifetime risk of 0.6% and an annual mortality of ~5,000. Despite the advent of immunochemotherapy and small molecule kinase inhibitors, there remains a great need for potent and safe treatment options for newly diagnosed, refractory, and relapsed CLL patients. In fact, there are currently no Food and Drug Administration (FDA)- approved therapeutic modalities in CLL that allow for selective targeting of malignant B cells without harming healthy cells and tissues. Notably, allogeneic hematopoietic stem cell transplantation (alloHSCT) has remained the only potentially curative treatment of CLL, underscoring the unmatched capability of immunotherapy. Pursuing potential contributors of the powerful graft-versus-leukemia (GVL) response, we devised a concerted antibody drug and target discovery strategy by generating the first post-alloHSCT antibody library from a cured CLL patient and selecting it by phage display against primary CLL cells. We identified a panel of fully human monoclonal antibodies (mAbs) with a common antigen on primary CLL cells. In still undisclosed work presented for the first time in this proposal, we identified the new target as Siglec-6. In healthy individuals, the expression of Siglec-6 is highly restricted to the placenta, to mast cells, and to certain B-cell subpopulations. Its expression on leukemia cells in CLL has not been reported previously. Our proposed study, which builds on our combined strengths in antibody engineering and transgenic mouse models, on the availability of clinical samples from both untreated and treated CLL patients for ex vivo and in vivo preclinical studies, as well as on our extensive supporting data, has the objective to rigorously test the hypothesis that Siglec-6 can serve as a target for T-cell recruiting and activating biAbs to mediate potent and safe eradication of leukemia cells in CLL patients. The experimental execution of this objective will be based on two Specific Aims. In Aim 1, we will build and test a panel of Siglec-6 x CD3 biAbs for primary CLL cell killing by autologous T-cells ex vivo and in vivo. In Aim 2, we will develop a novel CLL mouse model expressing transgenic human Siglec-6 and employ it for the in vivo evaluation of surrogate Siglec-6 x CD3 biAbs in a native and competent immune system. Collectively, our study explores a unique route for targeted therapy of CLL by interrogating a novel target with novel agents and a novel mouse model to assess and initiate further preclinical development.